Cell culture environments for the serum-free expansion of mesenchymal stem cells

ABSTRACT

Compositions and methods for promoting mesenchymal stem cell expansion while maintaining a pluripotent phenotype are disclosed. Serum-free cell culture systems and kits and methods of use for mesenchymal stem cell expansion are provided. Methods also comprise the use of the expanded mesenchymal stem cells to treat various disorders or diseases, particularly those of the cardiovascular system, bone, or cartilage.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/571,212, filed May 14, 2004, the content of which is herein incorporated by reference it its entirety.

FIELD OF THE INVENTION

The present invention relates to serum-free cell culture systems that provide for mesenchymal stem cell expansion while maintaining a pluripotent phenotype, and methods of use for the expanded mesenchymal stem cell populations.

BACKGROUND OF THE INVENTION

Mesenchymal stem cells (MSCs) are present in adult tissues and constitute a population of cells that can be isolated, expanded in culture, and characterized in vitro and in vivo (Pittenger and Martin (2004) Circ. Res. 95:9-20). MSCs are able to differentiate into multiple cell lineages, including osteoblasts, chondrocytes, endothelial cells, and neuronal cells, and can express phenotypic characteristics of endothelial, neural, smooth muscle, skeletal myoblast, and cardiac myocyte cells (Kassem et al. (2004) Basic Clin. Pharmacol. Toxicol. 95:209-214; Pittenger and Martin (2004) Circ. Res. 95:9-20). In recent years, MSCs have generated a high level of experimental and clinical interest due to their potential for a range of therapeutic uses including repair of damaged or diseased tissues (Baksh et al. (2004) J. Cell. Mol. Med. 8:301-316; Barry and Murphy (2004) Int. J. Biochem. Cell Bio. 36:568-584).

Typically, MSCs do very poorly in serum-free environments because they detach and die in culture. These MSCs can be maintained in an attached state in vitro with minimal serum (e.g., <1%), although such an environment provides little stimulation for MSCs to proliferate and grow. Although serum-free cell culture environments have been described for MSC expansion (Lennon et al. (1995) Exp. Cell Res. 219:211-222; U.S. Pat. No. 5,908,782), the current industry standard still contains a large amount of serum.

In addition to the desirability of serum-free environments for the expansion of MSCs, serum-free media and culture systems have great utility in the field of cellular therapy. The creation of highly defined environments for cell expansion is of great importance for quality purposes, and serum levels are typically very ill-defined (see, e.g., U.S. Pat. No. 5,908,782). In addition, there is a risk of Bovine Spongiform Encephalopathy (BSE) contamination in patients receiving cells cultured in the presence of serum. Such a risk raises the possibility that the FDA will not allow therapies involving cells cultured in the presence of animal sera.

For the aforementioned reasons, the development of new serum-free cell culture systems for the expansion of MSCs is therefore desirable.

SUMMARY OF THE INVENTION

Compositions and methods for promoting mesenchymal stem cell (MSC) expansion while maintaining a pluripotent phenotype are provided. The compositions include serum-free cell culture systems for MSC expansion that comprise a serum-free cell culture medium and a two-dimensional or three-dimensional cell culture surface. In the serum-free cell culture system of the present invention, at least one insoluble substrate protein is presented from the cell culture surface. In one embodiment, the cell culture surface comprises a cell culture support bound to a cell adhesion resistant (CAR) material, which in turn is bound to at least one insoluble substrate protein. Insoluble substrate proteins for use in the present invention include extracellular matrix (ECM) proteins such as fibronectin, laminin, hyaluronic acid (HA), vitronectin, collagen proteins such as collagen I, collagen II, collagen III, collagen IV, collagen V, and collagen VI, or any combination thereof. In one embodiment, the cell culture surface comprises a cell culture support bound to a cell adhesion resistant (CAR) material, which in turn is bound to at least one insoluble substrate protein, for example, at least one ECM protein. The serum-free cell culture medium is a solution that comprises a mixture of soluble MSC growth-promoting factors. Compositions further include kits comprising serum-free cell culture media and a two-dimensional or three-dimensional cell culture surface suitable for MSC expansion.

Methods of the present invention comprise the use of these serum-free cell culture systems to promote the expansion of MSCs. Further methods comprise the use of these serum-free cell culture systems and expanded MSCs for cell transplantation or to engineer tissues to treat various disorders or diseases, including those of the cardiovascular system, muscle, ligament, bone, tendon, cartilage, nervous system, blood, immune system, liver, or pancreas. Further methods comprise the use of these serum-free cell culture systems to promote the expansion of MSCs within primary aspirates from whole bone marrow such that the MSCs are co-cultured with non-MSCs present in the primary aspirates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows expansion of total mesenchymal stem cells (MSCs) over time in the G2 serum-free culture system and collagen 1+fibronectin surface of the present invention (BDT Environment) versus expansion in Cambrex Biosciences (Baltimore, Md.) serum-containing medium on tissue culture plastic.

FIG. 2 shows the adipocyte differentiation capacity of MSCs expanded in the G2 serum-free medium and collagen 1+fibronectin surface (BDT Environment) or expanded in Cambrex Biosciences (Baltimore, Md.) serum-containing medium on tissue culture plastic.

FIG. 3 shows the bone differentiation capacity of MSCs expanded in the G2 serum-free medium collagen 1+fibronectin surfaces (BDT Environment) or expanded in Cambrex Biosciences (Baltimore, Md.) serum-containing medium on tissue culture plastic.

FIG. 4 shows a comparison of the number of hMSCs after culture in a serum-free base medium (BDTM) with or without growth factors on either a tissue culture polystyrene (TCPS) or cell adhesion resistant (CAR) surface.

FIG. 5 shows a comparison of various serum-free compositions and collagen 1+fibronectin surfaces for MSC expansion that are labeled C6, D3, C2, and G5, compared to G2 medium and a serum containing medium on tissue culture plastic (10% FBS CM).

FIG. 6A shows the adipocyte differentiation capacity of MSCs expanded in D3, C2, and G5 serum-free culture media and collagen 1/fibronectin surfaces or expanded in Cambrex Biosciences (Baltimore, Md.) serum-containing medium on tissue culture plastic (10% FBS CM). FIG. 6B shows the bone differentiation capacity of MSCs expanded in D3, C2, and G5 serum-free culture media and collagen 1/fibronectin surfaces or expanded in Cambrex Biosciences (Baltimore, Md.) serum-containing medium on tissue culture plastic (10% FBS CM).

FIG. 7 shows a comparison of further refined serum-free composition for MSC expansion labeled G7, G4, and C8 and cultured on collagen 1/fibronectin surfaces compared to expansion in Cambrex Biosciences (Baltimore, Md.) serum-containing medium (10% FBS CM).

FIG. 8 shows a comparison of MSC expansion in various media conditions and collagen 1/fibronectin surfaces, including G4 serum-free medium, G4+TGFβ, FGF only, EGF only, and TGFβ only, as compared to basal medium and Cambrex Biosciences (Baltimore, Md.) serum-containing medium on tissue culture plastic (10% FBS CM).

FIG. 9 shows a comparison of MSC expansion in various media conditions where selected growth factor combinations were added to basal medium, as compared to basal medium alone and Cambrex Biosciences (Baltimore, Md.) serum-containing medium on tissue culture plastic (TCPS). Growth factor combinations were bFGF+TGF-β, WNT-3a+bFGF, bFGF+EGF, WNT-3a+TGF-β, WNT-3a+EGF, EGF+TGF-β, WNT-3a alone, bFGF+EGF+TGF-β, WNT-3a+bFGF+EGF+TGF-β, and bFGF+EGF+TGF-β+BIO.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for promoting mesenchymal stem cell (MSC) expansion while maintaining the pluripotent phenotype of these cells. Serum-free cell culture systems for MSC expansion are provided. These cell culture systems comprise serum-free cell culture medium and a two-dimensional or three-dimensional cell culture surface. The serum-free cell culture medium is a solution that comprises a mixture of soluble MSC growth-promoting and self-renewal factors. In the serum-free cell culture system of the present invention, at least one insoluble substrate protein is presented from the cell culture surface. In one embodiment, the cell culture surface comprises a cell culture support bound to a cell adhesion resistant (CAR) material, which in turn is bound to at least one insoluble substrate protein. Insoluble substrate proteins may include but are not limited to extracellular matrix (ECM) proteins such as fibronectin, laminin, hyaluronic acid (HA), vitronectin, or collagen proteins such as collagen I, collagen II, collagen III, collagen IV, collagen V, and collagen VI, or any combination thereof. Other compositions of the present invention include kits comprising the serum-free cell culture systems of the invention, which are suitable for MSC expansion.

Methods of the present invention are directed to the use of these serum-free cell culture systems to promote the expansion of MSCs and to engineer tissues. The expanded MSCs of the present invention can be used to treat various disorders or diseases, particularly those of the cardiovascular system, muscle, ligament, bone, tendon, cartilage, nervous system, blood, immune system, liver, or pancreas.

The term “mesenchymal” is intended to mean such cells as bone marrow, endothelial cells, epithelial cell progenitors, cardiomyocytes, astrocytes, neurons, chondrocytes, osteoblasts, pancreatic cells, hepatocytes, and other cells of mesenchymal origin. As used herein, the term “mesenchymal stem cell” or “MSC” refers to a cell that gives rise to a cell of mesenchymal lineage.

The term “expanded” is intended to mean that the resultant cell population is derived from ex vivo culture of stem cells in media compositions comprising mixtures of cytokines, where the outgoing (cultured) number of cells exceeds the ingoing (non-cultured) number of cells. The term “expanded” is not to be construed or limited by any mechanism or theory of cellular origin and may comprise cells that originate de novo in culture.

The serum-free cell culture system of the present invention comprises a serum-free cell culture medium and a cell culture surface. In one embodiment, at least one insoluble substrate protein is presented from the cell culture surface, meaning that the insoluble substrate protein is bound, adsorbed, linked, attached or in some way associated with the cell culture surface. Suitable insoluble substrate proteins include the ECM proteins disclosed herein. In one such embodiment, the insoluble substrate protein presented from the cell culture surface is a combination of collagen I and fibronectin. In another embodiment, the cell culture surface comprises a cell culture support bound to a cell adhesion resistant material, which in turn is bound to at least one insoluble substrate protein. The cell culture support may be solid or porous, polymer, metal, glass, ceramic, or combinations thereof. Culturing of MSCs in the presence of this cell culture surface and the serum-free cell culture medium unexpectedly provides for expansion of these cells while maintaining their pluripotent phenotype.

As used herein, the term cell adhesion resistant (CAR) refers to a material that, when present on a surface, prevents, inhibits, or reduces the non-specific binding (adhesion) of cells, proteins, or polypeptides found on cell surfaces. CAR materials are resistant to adhesion of mammalian cells and also to microorganisms. CAR materials are sometimes referred to as non-fouling substrates, inert coatings, low affinity reagents, or non-adhesive coatings.

The cell culture support to which is bound the CAR material may be two- or three-dimensional, solid or porous, and may be constructed of any of a variety of materials, including natural polymers, synthetic polymers, hydrogels, metals, ceramics, and inorganic or organic-inorganic composites. The cell culture support may be shaped using methods such as, for example, solvent casting, compression molding, filament drawing, meshing, leaching, weaving, and coating.

Examples of suitable polymers and hydrogels include, but are not limited to, collagen, glycosaminoglycan (GAG)-based materials, alginate, hyaluronate poly(alpha esters) such as poly(lactate acid), poly(glycolic acid), polyorthoesters and polyanhydrides and their copolymers, cellulose ether, cellulose, cellulosic ester, fluorinated polyethylene, phenolic, poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether, polyestercarbonate, polyether, polyetheretherketone, polyetherimide, polyetherketone, polyethersulfone, polyethylene, polyfluoroolefin, polylmide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinylidene fluoride, regenerated cellulose, urea-formaldehyde, or copolymers or physical blends of these materials.

Further examples include synthetic polymers, such as polylactic acid (PLA), polyglycolic acid (PGA), poly(orthoester), polysebacic acid polymers (PSA), poly(lactic-co-glycolic) acid copolymers (PLGA), poly(lactic-co-sebacic) acid copolymers (PLSA), poly(glycolic-co-sebacic) acid copolymers (PGSA), hydrogels such as polyhydroxyethylmethacrylate (poly-HEMA) or polyethylene oxide-polypropylene oxide copolymer, polyethylegylcols (PEG), polyvinylalchols (PVA), polyvinylpyrrolidone (PVP) and polyhydroxyalkanoate (PHA). PHAs and their production are described in, for example, PCT Publication Nos. WO 99/14313, WO 99/32536 and WO 00/56376. Hybrid materials containing naturally derived and synthetic polymer materials (e.g., PGA and PLGA) may also be used. Non-limiting examples of such materials are disclosed in Chen et al. (2000) Advanced Materials 12:455-457.

Other polymers useful in the present invention include polymers or copolymers of caprolactones, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates and degradable urethanes, as well as copolymers of these with straight chain or branched, substituted or unsubstituted, alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy- or di-carboxylic acids. In addition, the biologically important amino acids with reactive side-chain groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine and cysteine, or their enantiomers, may be included in copolymers with any of the aforementioned materials.

In one embodiment, polymer surfaces are selected from the group consisting of polystyrene, polyethylene, polypropylene, polyethylene terephthalate, polytetrafluoroethylene, polylactide, and cellulose. Silicone polymers such as polydimethylsiloxane (PDMS) are also used.

Inorganic composites include, e.g., calcium phosphate ceramics, bioglasses and bioactive glass-ceramics, in particular composites combining calcium hydroxyapatite and silicon stabilized tricalcium phosphate. Among preferred cell culture supports are polystyrene (PS), polypropylene, polyethylene, polyethylene terephthalate, polytri- or tetra-fluoroethylene, polyhexafluoropropylene, polyvinyl chloride, polyvinylidine fluoride, polyactide, cellulose, glass, or a ceramic.

Any suitable CAR material, many of which are known to those skilled in the art, may be bound to the cell culture support. Typical CAR materials include hyaluronic acid (HA) or a derivative thereof, alginic acid (AA) or a derivative thereof, poly-HEMA, polyethylene glycol (PEG), glyme or a derivative thereof, polypropylacrylamide, polyisopropylacrylamide, or a combination of these compounds. In one embodiment, the CAR material is HA.

In some embodiments, one or more of a proteoglycan, a biglycan, a glycosaminoglycan, or Matrigel™ may be bound to the CAR material.

In other embodiments, proteins or other substances may be bound to the CAR material either covalently or non-covalently, but are preferably covalently bound. The proteins or other substances comprise, for example, ECM proteins or polycationic polymers. ECM proteins for use in the present invention may include fibronectin, laminin, hyaluronic acid (HA), vitronectin, or collagen proteins such as collagen I, collagen II, collagen III, collagen IV, collagen V, and collagen VI, or any combination thereof. Various types of covalent bonds can form, some of which are discussed in more detail in co-pending, commonly assigned U.S. Patent Application Publication Nos. 20050036980, 20040062882, 20030113813, and 20030113812, all of which are incorporated herein by reference. These applications also disclose other aspects of making and using surfaces that include cell culture supports with bound CAR materials and ECM proteins.

In one embodiment, one or more ECM proteins as disclosed herein and/or one or more polycationic polymer are bound to the CAR material. In one embodiment, a mixture of collagen I and fibronectin is bound to the CAR material.

The ECM protein(s) can be in the form of a naturally occurring polypeptide (protein), a recombinant polypeptide, or a synthetic or semi-synthetic polypeptide, including any fragment of the peptide or protein, or combination thereof. The terms “polypeptide” and “protein” are used interchangeably herein.

Methods of cloning, expressing, and purifying polypeptides, such as ECM proteins, are conventional, as are methods of generating synthetic or semi-synthetic polypeptides. ECM proteins can also be obtained from commercial sources.

ECM proteins or polycationic polymers can be bound to the CAR material either covalently or non-covalently (for example, passively adsorbed, such as by electrostatic forces, ionic or hydrogen bonds, hydrophilic or hydrophobic interactions, Van der Waals forces, etc.). In a preferred embodiment, the binding is covalent. U.S. Patent Application Publication Nos. 20040062882, 20030113813, and 20030113812 describe such covalent binding of molecules to CAR surfaces.

Methods of making surfaces in which a CAR material is bound to a cell culture support, and in which ECM proteins, polycationic polymers, or the like, are bound to the CAR material, are described in detail in U.S. Patent Application Publication Nos. 20040062882, 20030113813, and 20030113812. In brief, one method of attaching a CAR material to a cell culture support comprises treating the cell culture support with an oxidizing plasma, and binding the CAR material to the treated cell culture support. Another method of attaching a CAR material to a cell culture support comprises treating the cell culture support with an oxidizing plasma; exposing the treated cell culture support to a polycationic polymer with amino groups (such as PEI, PDL, poly-L-lysine (PLL), poly-L-ornithine (PLO), poly-D-ornithine (PDO), poly(vinylamine) (PVA) or poly(allylamine) (PAA), in one embodiment, PEI or PDL to form an intermediate layer, and binding the CAR material to the intermediate layer. Methods of binding an ECM or a polycationic polyaminoacid to a CAR material are conventional. These include, for example, sodium periodate oxidation and reductive amination, EDC/NHS carbodiimide coupling, or the like.

This invention also speaks to the use of flexible substrates in culture. For example, Flexercell culture systems from Flexcell International Corporation are able to apply tensile, compressive, or shear stresses to cultured cells. See, for example, U.S. Pat. Nos. 4,789,601, 4,822,741, 4,839,280, 6,037,141, 6,048,723, and 6,218,178. U.S. Pat. No. 6,057,150 discloses the application of a biaxial strain to an elastic membrane that may be coated with extracellular matrix proteins and covered with cultured cells. U.S. Pat. No. 6,107,081 discloses another system in which a unidirectional cell stretching device comprising an elastic strip is coated with an extracellular matrix on which cells are cultured and stretched. A flexible substrate can be deformed easily and in a controlled manner, and also supports cell adhesion and growth comparable to conventional cell culture substrates. Silicones, such as poly(dimethyl siloxane) (PDMS), are particularly suitable for this application because they are not only highly flexible but also provide optical clarity that allows microscopic observation of the cell cultures.

Another aspect of the invention is a method of making the above cell culture surface of the invention, where the method comprises (a) attaching a CAR material to a cell culture support, and (b) attaching to the CAR material collagen I and fibronectin, or biologically active fragment or variant thereof, and, optionally, one or more other ECM proteins (or a biologically active fragment or variant of the ECM protein) and/or one or more polycationic polymers. Any of the ECM proteins or polycationic polymers disclosed herein, or others, may be used.

In one embodiment, the CAR material is attached to the cell culture support by treating the cell culture support with an oxidizing plasma, and binding the CAR material to the treated cell culture support. In another embodiment, the CAR material is attached to the cell culture support by treating the cell culture support with an oxidizing plasma; exposing the treated cell culture support to a polycationic polymer with amino groups to form an intermediate layer; and binding the CAR material to the intermediate layer. Preferably, the polycationic polymer is polyethylene imine (PEI) or poly-D-lysine (PDL).

In a one embodiment, a CAR material, such as HA, is bonded directly to polymeric nitrogen-containing surfaces. Examples of such surfaces are ammonia plasma-treated polymers and Primaria ™-treated polystyrene (PS) surfaces, and poly D-lysine coated surfaces. Polymeric substrates suitable for use in the invention include polystyrene, polypropylene, polyethylene terephthalate, polylactide, cellulose, and the like, though polystyrene is preferred.

HA is immobilized directly on an intermediate polyamine layer, e.g., polyethyleneimine, poly-D-Lysine, or poly-L-lysine, or directly bound to the polymer with the HA bound to that intermediate layer).

The use of plasma techniques are familiar to those of skill in the art (see, for example, Garbassi et al., (1994) Polymer Surfaces, from Physics to Technology (Wiley, Chichester), and Inagaki (1996) Plasma Surface Modification and Plasma Polymerization (Technomic Publishing Company, Lancaster). In the present invention, the plasma treatment process may be any process that is capable of causing nitrogen to be incorporated onto the surface of the polymer article resulting in reactive amine or other nitrogen-containing groups, including direct as well as remote plasma treatment methods. Examples of suitable plasma treatments are ones using reactive gases such as nitrogen, nitrogen oxide, nitrogen dioxide or ammonia in the gas phase, alone or in mixture with air, argon, or other inert gases, and may be preceded or followed by treatments employing argon or other inert gases. The plasma maybe sustained over the full treatment time or maybe administered in pulses. Preferably, the plasma gas is ammonia, and treatment is performed with a power charge of between 1 and 400 W, preferably between 10 and 150 W, a pressure between 10 mtorr and 10 ton, and a treatment time between 1 second and 1 hour, preferably between 10 seconds and 30 minutes.

Plasma-treated polystyrene can be prepared, for example by pumping the treatment chamber to a 0.3 mTorr base pressure, establishing a 200 mTorr argon atmosphere, and applying a 60 sec argon plasma treatment, followed by a 120 sec, 375 mTorr NH₃ plasma treatment at 95 W. Other suitable treatments will be known to those of skill in the art. Following plasma treatment of the surface to be coated, the plasma-treated surface may be exposed to an aqueous solution containing HA or a derivative thereof, or alginic acid (alginate; AA) in the presence of a condensing agent such as a carbodiimide, for example, ethyldimethylaminopropyl carbodiimide (EDC), in aqueous solution, or dicyclohexylcarbodiimide (DCC), in organic solvents. The term “expose” or “exposing” as used herein is intended to include any type of contact made between a liquid and a solid, for example by pipetting, pouring, spraying, dripping, immersing, pouring, dipping, injecting, etc., without limitation.

HA is an anionic polysaccharide composed of repeating units of beta-1,4-glucuronatebeta-1,3-N-acetylglucosamine. A reactive —COO⁻ group is present on every repeat unit of HA that can be utilized to covalently couple HA to an amine containing surface using methods described herein. In this manner, a condensing agent such as EDC activates the —COO⁻ groups present in HA, creating a reactive ester intermediate (ester (o-acylisourea) intermediate). This intermediate is highly unstable and subject to hydrolysis, leading to the cleaving off of the activated ester intermediate, forming an isourea, and regenerating the —COO⁻ group. To stabilize this reactive ester intermediate, and increase reaction yield, a molecule able to enhance the reaction promoted by EDC should also be present. Such stabilizing compounds are generally selected from the class of N-hydroxysuccinimides and aryl or heterocyclic derivatives thereof. Preferred N-hydroxysuccinimides include, but are not limited to, N-hydroxy-succinimide (NHS), hydroxy-sulfosuccinimide (sulfo-NHS) or hydroxybenzotriazole hydrate. Although not intended to be bound to a particular theory, it is believed that attachment of HA to the amine containing polymer surface occurs through a mechanism wherein (for example) EDC and NHS combine to create an active ester polysaccharide with a carboxyl group capable of coupling to an amine. When coupling occurs, NHS is released. Other compounds known in the art that are able to react with EDC in this manner and which serve as reactive intermediate ester stabilizing compounds should also be effective in the invention. HA covalently bonded to plasma-treated polystyrene in this way prevents attachment of any of a number of types of cells, including NIH3T3 and osteoblast MC3T3 cells as well as MSC's and a variety of primary and stem cells.

Suitable derivatives of HA that may be used in the invention will be known to the skilled artisan, and are described, for example, in U.S. Pat. No. 4,851,521. These include partial esters of HA with alcohols of the aliphatic, araliphatic, cycloaliphatic and heterocyclic series, and salts of such partial esters with inorganic or organic bases. Similar derivatives of alginic acid should also be useful. Further, other plasma treatment methods for producing surfaces with amine and other nitrogen-containing groups are also suitable, and are known to those of skill in the art.

The cell culture surfaces for use in the serum-free cell culture systems include but are not limited to standard tissue culture vessels and two-dimensional surfaces, including sheets, slides, culture dishes, culture flasks, bags, culture bottles, or multiwell dishes. Alternatively, three-dimensional cell culture surfaces, such as microcarriers or three dimensional (3-D) scaffolds, including but not limited to foams, hydrogels, or fiber meshes may be used for generating a three-dimensional cell culture, tissue, or organ. “Three-dimensional scaffold” refers herein to a 3-D porous template that provides a very high surface area to volume ratio for cell culture. These scaffolds may be used for initial cell attachment and expansion, or subsequently for tissue formation either in vitro or in vivo. A 3-D scaffold according to this invention comprises base materials (described below), a CAR layer and bound thereto one or more insoluble substrate proteins, such as the ECMs disclosed herein, and, optionally, other substances, which promote or enhance cell attachment, growth, migration, and/or differentiation. In one embodiment, the scaffold is seeded with MSCs and contacted with the serum-free cell culture medium described herein below in order to permit cell growth and differentiation in a structural environment that more closely mimics the in vivo setting. Cells derived therefrom can be isolated from the scaffold or can be implanted with or without the scaffold into a suitable location in the body of a mammal, preferably a human patient. However, the use of this technology could easily be translated to non-human mammals such as cats, dogs, horses, and the like.

The shape and dimensions of the 3-D scaffold are determined based on the organ being replaced or supplemented, and the type of scaffold material being used to create the construct. For example, if a polymeric scaffold is used for heart tissue replacement or supplementation, the dimension of the polymeric scaffold can vary in terms of width and length of the polymeric scaffold. One of skill in the art recognizes that the size and dimensions of the polymeric scaffold will be determined based on the area of the organ being replaced or supplemented. Furthermore, other suitable articles may constitute a cell culture support surface of the present invention, including medical devices, extracorporeal devices and artificial joints, tubes, sutures, stents, orthopedic devices, vascular grafts, membranes, films, biosensors, or microparticles.

One embodiment includes a method of forming tissue-engineered constructs using a 3-D scaffold material or other suitable article that comprises the CAR surface onto which one or more insoluble substrate proteins, for example, the ECMs disclosed herein, are attached, either covalently or non-covalently. Such a scaffold in combination with the serum-free cell culture medium disclosed herein supports the maturation, development and differentiation, of additional cultured MSCs in vitro to form components of adult tissues analogous to their in vivo counterparts.

The tissue-engineered constructs, in one embodiment, are created using scaffold materials disclosed herein as the substrate onto which cells are deposited and cultured in the presence of the serum-free cell culture medium described herein below, and on which cells are grown and adhere. The scaffold allows optimum cell-cell interactions, thereby allowing a more natural formation of cellular phenotypes and a tissue microenvironment. The scaffold also allows MSCs to continue to grow actively, proliferate, and differentiate to produce a tissue-engineered construct that is also capable of supporting the growth, proliferation, and differentiation of additional cultured cell populations, if needed.

In one embodiment, the scaffold is biocompatible and conducive to cell attachment and subsequent tissue growth. Other surface properties can be modified to suit the intended application without altering other properties of the scaffold such as its mechanical strength or thermal properties. Useful surface modifications could include, for example, changes in chemical group functionality, surface charge, hydrophobicity, hydrophilicity, and wettability. The CAR surface technology can easily be translated to the surfaces of 3-D scaffolds. Such surface modifications are well known in the art. Sterilization is performed prior to seeding the scaffold with cells. Heat sterilization is often impractical since the heat treatment could deform the device, especially if the materials have a melting temperature below that required for the heat sterilization treatment. For example, cold ethylene oxide gas, vapor hydrogen peroxide treatments can be used for sterilization.

The 3-D scaffolds of the present invention comprise any suitable base material for construction, including the polymeric materials disclosed herein above. The polymeric matrix can be fabricated to have a controlled pore structure that allows nutrients from the serum-free cell culture medium described herein below to reach the deposited cell population but prevent cultured cells from migrating through the pores. In vitro cell attachment and cell viability can be assessed using scanning electron microscopy, histology and quantitative assessment with radioisotopes.

The polymeric matrix can be shaped into any number of desirable configurations to satisfy any number of overall system, geometry, or space restrictions. The polymeric matrix can be shaped to different sizes to conform to the organs of different sized patients. Thus, the tissue-engineered construct can be flat, tubular, or of complex geometry. The shape of the construct will be decided by its intended use. The construct can be implanted to repair, supplement, or replace diseased or damaged parts of organs.

In one embodiment, the scaffold base material is a hydrogel composed of crosslinked polymer networks that are typically insoluble or poorly soluble in water, but can swell to an equilibrium size in the presence of excess water. For example, the MSCs can be placed in a hydrogel and the hydrogel injected into desired locations within the organ. The hydrogel compositions can include, without limitation, for example, poly(esters), poly(hydroxy acids), poly(lactones), poly(amides), poly(ester-amides), poly(amino acids), poly(anhydrides), poly(ortho-esters), poly(carbonates), poly(phosphazines), poly(thioesters), polysaccharides and mixtures thereof. Furthermore, the compositions can also include, for example, a poly(hydroxy) acid including poly(alpha-hydroxy) acids and poly(betahydroxy) acids. Such poly(hydroxy) acids include, for example, polylactic acid, polyglycolic acid, polycaproic acid, polybutyric acid, polyvaleric acid, and copolymers and mixtures thereof. Due to the unique properties of hydrogels and their potential applications in such areas as controlled drug delivery, various types of hydrogels have been synthesized and characterized. The matrix materials of the present invention encompass both conventional foam or sponge materials and the so-called hydrogel sponges (see, e.g., U.S. Pat. No. 5,451,613).

Mesenchymal stem cells grown on the scaffold materials in the presence of the serum-free cell culture medium disclosed herein in accordance with the present invention may grow in multiple layers, forming a cellular structure that resembles physiologic conditions found in vivo. The scaffold can support the proliferation of different types of cells and the formation of a number of different tissues. Examples include, but are not limited to, kidney, heart, skin, liver, pancreas, adrenal and neurological tissue, as well as tissues of the gastrointestinal and genitourinary tracts, and the circulatory system.

The cells grown or expanded on the aforementioned scaffold can be used, alone or in combination with the scaffold, in a variety of applications. For example, the scaffold and cells can be implanted into a subject. Implants can be used to replace or supplement existing tissue, for example, by treating a subject with a cardiovascular disorder by replacing or supplementing natural cardiovascular tissue. The subject can be monitored after implantation for amelioration of the cardiovascular disorder. A three-dimensional biocompatible scaffold may be brought into contact with vasculature-promoting expanded MSCs of the invention and then brought into contact with a host tissue at a target site (e.g., within the organ) or where the organ tissue is grown on the scaffold prior to implantation. The graft is then able to grow and proliferate within the target site and replace or supplement the depleted activity of the organ. The construct can be added at a single location in the host or, alternatively, a plurality of constructs can be created and added to multiple sites in the host.

The term “target site” as used herein refers to a region in the host or organ that requires replacement or supplementation. The target site can be a single region in the organ or host, or can be multiple regions in the organ or host. In some embodiments, the supplementation or replacement results in the same physiological response as a normal organ.

In another embodiment, the scaffold is created using parts of a natural decellularized organ. Parts of organs can be decellularized by removing the entire cellular and tissue content from the organ (see, e.g., U.S. Pat. No. 6,479,064). The term “decellularized” or “decellularization” as used herein refers to a biostructure (e.g., an organ, or part of an organ) from which the cellular and tissue content has been removed leaving behind an intact acellular infrastructure. The process of decellularization removes the specialized tissue, leaving behind the complex three-dimensional network of connective tissue. The connective tissue infrastructure is primarily composed of collagen. The decellularized structure provides a matrix material onto which different cell populations can be infused. Decellularized biostructures can be rigid, or semi-rigid, having an ability to alter their shapes. Culture and construction of decellularized biostructures can be performed, for example, as describe in U.S. Pat. No. 6,479,064, which is herein incorporated by reference in its entirety.

The two-dimensional and three-dimensional cell culture surfaces serve as a substrate onto which MSCs can be seeded and cultured in the presence of a serum-free cell culture medium disclosed herein. In this manner, seeded MSCs can be expanded ex vivo by contacting them with a serum-free cell culture medium that comprises a defined cytokine cocktail. In one embodiment, the defined combinations comprise at least 2, at least 3, at least, 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 cytokines from the group comprising brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), bone morphogenic protein 2 (BMP-2), bone morphogenic protein 4 (BMP-4), dickkopf 1 (DKK-1), epidermal growth factor (EGF), erythropoietin (EPO), fibronectin, Flt-3/Flk-2 ligand, granulocyte colony stimulating factor (G-CSF), insulin-like growth factor (IGF-1), interleukin-11 (IL-11), interleukin-3 (IL-3), interleukin-5 (IL-5), interleukin-6 (IL-6), leukemia inhibitory factor (LIF), platelet-derived endothelial cell growth factor (PD-ECGF), stem cell factor (SCF), stromal cell derived factor 1-α (SDF 1-α), transforming growth factor β (TGF-β), thrombospondin, and a WNT signaling agonist. WNT signaling agonists include, but are not limited to (2′Z,3′E)-6-bromoindirubin-3′-oxime (BIO) and WNT proteins including WNT-1, WNT-2, WNT-2b, WNT-3, WNT-3a, WNT-4, WNT-5a, WNT-5b, WNT-6, WNT-7a, WNT-7b, WNT-8a, WNT-8b, WNT-9a, WNT-9b, WNT-10a, WNT-10b, WNT-11, and WNT-16. In one embodiment, the WNT protein is WNT-3a.

In one embodiment, the defined cytokine cocktail is termed “G2” and comprises a combination of fibronectin, SDF-11, IL-6, SCF, IL-5, BDNF, PD-ECGF, IL-11, IL-3, EPO, Flt-3/Flk-2 ligand, BMP-4, thrombospondin, IGF-1, and bFGF.

In another embodiment, the defined cytokine cocktail is termed “C6” and comprises a combination of BDNF, bFGF, BIO, BMP-2, BMP-4, DKK-1, EGF, EPO, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, G-CSF, IGF-1, IL-11, IL-3, IL-5, IL-6, LIF, PD-ECGF, SCF, SDF-1α, and WNT-3a.

In another embodiment, the defined cytokine cocktail is termed “D3” and comprises a combination of bFGF, BMP-2, EGF, EPO, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and WNT-3a.

In another embodiment, the defined cytokine cocktail is termed “C2” and comprises a combination of bFGF, BIO, BMP-2, BMP-4, EGF, EPO, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and WNT-3a.

In another embodiment, the defined cytokine cocktail is termed “G5” and comprises a combination of bFGF, BMP-4, DKK-1, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-6, PD-ECGF, SDF-1α, and WNT-3a.

In another embodiment, the defined cytokine cocktail is termed “G7” and comprises a combination of bFGF, BMP-2, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, LIF, and WNT-3a.

In another embodiment, the defined cytokine cocktail is termed “G4” and comprises a combination of bFGF, BMP-2, EGF, IL-11, PD-ECGF, and WNT-3a.

In another embodiment, the defined cytokine cocktail is termed “C8” and comprises a combination of bFGF, BMP-2, EGF, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, IL-5, LIF, PD-ECGF, and WNT-3a.

In another embodiment, the cytokine cocktail comprises bFGF in combination with at least growth factor selected from the group consisting of a WNT signaling agonist, TGF-β, and EGF. In another embodiment, the cytokine cocktail comprises a WNT signaling agonist in combination with at least one growth factor selected from the group consisting of bFGF, TGF-β, and EGF. In another embodiment, the cytokine cocktail comprises TGF-β in combination with at least one growth factor selected from the group consisting of bFGF, a WNT signaling agonist, and EGF. In another embodiment, the cytokine cocktail comprises EGF in combination with at least one growth factor selected from the group consisting of bFGF, a WNT signaling agonist, and TGF-β. In another embodiment, the cytokine cocktail comprises at least two growth factors selected from the group consisting of bFGF, TGF-β, EGF, and a WNT signaling agonist. In some of these embodiments, the WNT signaling agonist is WNT-3a; in other of these embodiments, the WNT signaling agonist is BIO.

In one embodiment, adding complexity to a media composition comprising at least two growth factors selected from the group consisting of bFGF, TGF-β, EGF, and a WNT signaling agonist with the addition of other factors enhances MSC expansion. For example, although media containing combinations of bFGF, TGF-β, EGF, and a WNT signaling agonist produce MSC expansion, the addition of other signaling molecules such as BMPs, soluble ECMs, and other cytokines and factors can further enhance MSC expansion.

The G2 combination of growth factors allows for rapid expansion of MSCs, at similar or even better than commercially available serum containing media. In particular, this expansion, when compared to serum containing media that is greater than 10 days old, will outperform the serum containing media. Furthermore, the MSCs expanded in G2 media are still pluripotent and have been demonstrated to differentiate into both bone and fat. The ability of these MSCs to maintain a pluripotent phenotype is significant as this is evidence that serum can be removed from the culture during expansion and the cells have not changed considerably nor have they been pushed towards specific differentiated phenotypes that would prevent the use of the expanded cells from being used in different therapeutic applications.

The defined cytokine combinations are added to a serum-free base medium to provide a serum-free culture medium suitable for expanding MSCs. Any serum-free nutritive medium suitable for the culture of MSCs may be used. In one embodiment, the base medium is as described in Lennon et al. (1995) Exp. Cell Res., 219:211-222, comprising Dulbecco's Modified Eagle's Medium (DMEM), MCDB-201 (Sigma-Aldrich (St. Louis, Mo.)), ascorbic acid 2-phosphate, dexamethasone, linoleic acid-bovine serum albumin, insulin, transferrin, and sodium selenite. In a further embodiment, the base medium comprises a ratio of DMEM to MCDB-201 of 60:40, the concentration of ascorbic acid 2-phosphate is 1×10⁴ M, the concentration of dexamethasone is 1×10⁻⁹ M, linoleic acid-bovine serum albumin is in an amount of about 0.1%, insulin is in an amount of about 5 μg/mL, transferrin is in an amount of about 5 μg/mL, and sodium selenite is in an amount of about 5 ng/mL (see Table 2, herein below). Many different base media could be used, and human or other animal-derived components may be substituted such as human albumin for bovine albumin, or human insulin. The ability to substitute non-animal derived components for animal derived is known in the field, and several of these components are commercially available, for example from Cambrex Biosciences (Baltimore, Md.) or Sigma-Aldrich (St. Louis, Mo.).

The final concentration of cytokines in the serum-free cell culture medium can range from about 1 femtogram/ml to about 1 picogram/ml to about 1 nanogram/ml to about 1 microgram/ml to about 1 milligram/ml. In some embodiments, the concentration of any one of the cytokines can be about 1 pg/ml, about 5 pg/ml, about 10 pg/ml, about 15 pg/ml, about 20 pg/ml, about 25 pg/ml, about 30 pg/ml, about 35 pg/ml, about 40 pg/ml, about 45 pg/ml, about 50 pg/ml, about 55 pg/ml, about 60 pg/ml, about 65 pg/ml, about 70 pg/ml, about 75 pg/ml, about 80 pg/ml, about 85 pg/ml, about 90 pg/ml, about 95 pg/ml, about 100 pg/ml, about 110 pg/ml, about 120 pg/ml, about 130 pg/ml, about 140 pg/ml, about 150 pg/ml, about 160 pg/ml, about 170 pg/ml, about 180 pg/ml, about 190 pg/ml, about 200 pg/ml, about 210 pg/ml, about 220 pg/ml, about 230 pg/ml, about 240 pg/ml, about 250 pg/ml, about 260 pg/ml, about 270 pg/ml, about 280 pg/ml, about 290 pg/ml, about 300 pg/ml, about 310 pg/ml, about 320 pg/ml, about 330 pg/ml, about 340 pg/ml, about 350 pg/ml, about 360 pg/ml, about 370 pg/ml, about 380 pg/ml, about 390 pg/ml, about 400 pg/ml, about 410 pg/ml, about 420 pg/ml, about 430 pg/ml, about 440 pg/ml, about 450 pg/ml, about 460 pg/ml, about 470 pg/ml, about 480 pg/ml, about 490 pg/ml, about 500 pg/ml, about 510 pg/ml, about 520 pg/ml, about 530 pg/ml, about 540 pg/ml, about 550 pg/ml, about 560 pg/ml, about 570 pg/ml, about 580 pg/ml, about 590 pg/ml, about 600 pg/ml, about 610 pg/ml, about 620 pg/ml, about 630 pg/ml, about 640 pg/ml, about 650 pg/ml, about 660 pg/ml, about 670 pg/ml, about 680 pg/ml, about 690 pg/ml, about 700 pg/ml, about 710 pg/ml, about 720 pg/ml, about 730 pg/ml, about 740 pg/ml, about 750 pg/ml, about 760 pg/ml, about 770 pg/ml, about 780 pg/ml, about 790 pg/ml, about 800 pg/ml, about 810 pg/ml, about 820 pg/ml, about 830 pg/ml, about 840 pg/ml, about 850 pg/ml, about 860 pg/ml, about 870 pg/ml, about 880 pg/ml, about 890 pg/ml, about 900 pg/ml, about 910 pg/ml, about 920 pg/ml, about 930 pg/ml, about 940 pg/ml, about 950 pg/ml, about 960 pg/ml, about 970 pg/ml, about 980 pg/ml, about 990 pg/ml, about 1 ng/ml, about 1.5 ng/ml, about 2 ng/ml, about 2.5 ng/ml, about 3 ng/ml, about 3.5 ng/ml, about 4 ng/ml, about 4.5 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, about 15 ng/ml, about 20 ng/ml, about 25 ng/ml, about 30 ng/ml, about 35 ng/ml, about 40 ng/ml, about 45 ng/ml, about 50 ng/ml, about 55 ng/ml, about 60 ng/ml, about 65 ng/ml, about 70 ng/ml, about 75 ng/ml, about 80 ng/ml, about 85 ng/ml, about 90 ng/ml, about 95 ng/ml, about 100 ng/ml, about 110 ng/ml, about 120 ng/ml, about 130 ng/ml, about 140 ng/ml, about 150 ng/ml, about 160 ng/ml, about 170 ng/ml, about 180 ng/ml, about 190 ng/ml, about 200 ng/ml, about 210 ng/ml, about 220 ng/ml, about 230 ng/ml, about 240 ng/ml, about 250 ng/ml, about 260 ng/ml, about 270 ng/ml, about 280 ng/ml, about 290 ng/ml, about 300 ng/ml, about 310 ng/ml, about 320 ng/ml, about 330 ng/ml, about 340 ng/ml, about 350 ng/ml, about 360 ng/ml, about 370 ng/ml, about 380 ng/ml, about 390 ng/ml, about 400 ng/ml, about 410 ng/ml, about 420 ng/ml, about 430 ng/ml, about 440 ng/ml, about 450 ng/ml, about 460 ng/ml, about 470 ng/ml, about 480 ng/ml, about 490 ng/ml, about 500 ng/ml, about 510 ng/ml, about 520 ng/ml, about 530 ng/ml, about 540 ng/ml, about 550 ng/ml, about 560 ng/ml, about 570 ng/ml, about 580 ng/ml, about 590 ng/ml, about 600 ng/ml, about 610 ng/ml, about 620 ng/ml, about 630 ng/ml, about 640 ng/ml, about 650 ng/ml, about 660 ng/ml, about 670 ng/ml, about 680 ng/ml, about 690 ng/ml, about 700 ng/ml, about 710 ng/ml, about 720 ng/ml, about 730 ng/ml, about 740 ng/ml, about 750 ng/ml, about 760 ng/ml, about 770 ng/ml, about 780 ng/ml, about 790 ng/ml, about 800 ng/ml, about 810 ng/ml, about 820 ng/ml, about 830 ng/ml, about 840 ng/ml, about 850 ng/ml, about 860 ng/ml, about 870 ng/ml, about 880 ng/ml, about 890 ng/ml, about 900 ng/ml, about 910 ng/ml, about 920 ng/ml, about 930 ng/ml, about 940 ng/ml, about 950 ng/ml, about 960 ng/ml, about 970 ng/ml, about 980 ng/ml, about 990 ng/ml, or about 1000 ng/ml.

In one embodiment, the serum-free cell culture medium comprises a concentration of BDNF from about 10 pg/ml to about 500 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of bFGF from about 1 pg/ml to about 500 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of BIO of about 1 pM to about 1 pM. In another embodiment, the serum-free cell culture medium comprises a concentration of BMP-2 from about 10 pg/ml to about 500 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of BMP-4 from about 10 pg/ml to about 500 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of DKK-1 from about 10 pg/ml to about 10 μg/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of EGF from about 10 pg/ml to about 100 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of erythropoietin from about 0.0001 units/ml to about 50 units/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of fibronectin from about 10 pg/ml to about 100 μg/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of Flt-3/Flk-2 ligand from about 1 pg/ml to about 100 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of G-CSF from about 1 pg/ml to about 100 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of IGF-1 from about 10 pg/ml to about 500 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of IL-11 from about 1 pg/ml to about 100 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of IL-3 from about 1 pg/ml to about 100 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of IL-5 from about 1 pg/ml to about 100 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of IL-6 from about 1 pg/ml to about 100 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of LIF from about 10 pg/ml to about 100 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of PD-ECGF from about 10 pg/ml to about 500 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of SCF from about 10 pg/ml to about 100 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of SDF-1α from about 1 pg/ml to about 100 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of TGF-β from about 10 fg/ml to about 100 ng/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of thrombospondin from about 10 pg/ml to about 1 μg/ml. In another embodiment, the serum-free cell culture medium comprises a concentration of WNT-3a from about 10 pg/ml to about 500 ng/ml.

In one embodiment, the serum-free cell culture medium comprises BDNF in an amount of about 1 ng/ml; bFGF in an amount of about 2.5 ng/ml; BMP-4 in an amount of about 0.5 ng/ml; erythropoietin in an amount of about 0.05 U/ml; fibronectin in an amount of about 10 ng/ml; Flt-3/Flk-2 ligand in an amount of about 5 ng/ml; IGF-1 in an amount of about 2.5 ng/ml; IL-11 in an amount of about 0.1 ng/ml; IL-3 in an amount of about 1 ng/ml; IL-5 in an amount of about 0.1 ng/ml; IL-6 in an amount of about 0.2 ng/ml; PD-ECGF in an amount of about 2 ng/ml; SCF in an amount of about 2 ng/ml; SDF-1α in an amount of about 3 ng/ml; and thrombospondin in an amount of about 10 ng/ml.

Those skilled in the art will recognize that cytokines for use in the present invention may be concentrated and, in some instances, lyophilized before addition to the base medium to obtain the examples of final concentrations listed above. Those skilled in the art will also recognize that the mass weight added to culture will depend on the specific biological activity of the cytokine preparation. Bioassays to determine the biological potency of cytokines are well known in the art. Therefore, where the biological activity is correlated to a mass weight, then biological “units” as defined by the assay are used.

Populations of MSCs that have been expanded using the serum-free cell culture system of the present invention comprise cells expressing cell surface markers of interest. The term “cell surface marker” or “marker” is intended to mean a protein that is expressed on the surface of a cell, which can be detected using specific antibodies. For example, the expanded MSCs of the present invention express markers reported for commercial MSCs, including CD166, CD44, CD105, and CD29. The expanded MSCs of the invention may also express combinations of these cell markers, and may express CD73, CD90, CD 106, CD 146, or any combination thereof. In some instances, the expression of a cell surface marker defines non-mesenchymal cell populations such as mature blood cells and hematopoietic stem cells, for example: CD3; CD14; CD19; CD34; CD42a; CD45; and any combinations of these markers.

The term “substantially free” is intended to mean that less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the cells within the population express the marker of interest. For example, in one embodiment, the expanded cell population is substantially free of T-cells (expressing the CD3 antigen), B-cells (expressing the CD 19 antigen), or mature granulocytes, NK lymphocytes, or macrophages (expressing the CD16 antigen).

The term “substantial proportion” is intended to mean that at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or up to 100% of the cells express the marker of interest. Conversely, the term “substantially diminished” is intended to mean that at least less than 10%, at least less than 5%, or at least less than 1% of cells express the marker of interest.

Those skilled in the art recognize that cells with increased cell surface marker expression can be detected and isolated by any means including flow cytometric sorting, antibody panning, and the like. Generally in flow cytometric analysis, one of skill in the art must first set a detection threshold for fluorescence. In setting the threshold, a negative control sample population will be recorded and a gate will be set around the population of interest according to the desired forward scatter (FSC) and side scatter (SSC). The detection threshold is then adjusted so that 97% or more of the cells do not fluoresce. Once the detection threshold is set, the fluorescence of cell population of interest is recorded. A cell is considered “positive for expression” when it expresses the marker of interest, whether a protein or a gene. Any method may be used to determine expression such as gene expression profiles, FACS, and the like. The term “⁺” indicates that the cell is positive for expression of the marker of interest. The term “⁻” indicates that the cell does not have detectable levels of expression of the marker of interest.

The antibodies used to detect various lineages may be conjugated to different fluorochromes. These include phycobiliproteins, e.g., phycoerythrin and allophycocyanins; fluorescein; and Texas red. Dead cells can also be detected using dyes that selectively accumulate in dead cells (e.g., propidium iodide and 7-amino actinomycin D).

These expanded MSCs are characterized by a capacity for self-renewal. Further, the cells are characterized by an ability to commit to multilineage development. By “multilineage development” is intended that the cell is capable of differentiating into cell types of mesenchymal origin. These cells have a capability of limited self renewal and are constrained to committed development to various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma (Pittenger et al. (1999) Science. 284:5411 143).

Stem cell function can be assayed using both in vitro and in vivo methods. In vitro testing comprises culturing MSCs in semi-solid medium similar to hematopoetic stem cells. Along with colony forming assays, MSCs may be tested for multilineage potential by culturing these cells in the presence of factors that send them down specific pathways such as bone, fat, and cartilage. Media for differentiating expanded MSCs are well known in the art (Pittenger and Martin (2004) Circ. Res. 95:9-20), and are commercially available through several sources such as Cambrex (Baltimore, Md.), Stem Cell Technologies (Vancouver, B. C., Canada), and R&D Systems (Minneapolis, Minn.). Expanded cells are cultured either on surfaces such as culture dishes with these media for 7-30 days for bone, fat, or muscle phenotypes, or in pellets in the bottom or tubes or flasks for differentiating down cartilage phenotypes. The test for function, tissue specific tests are performed such as alkaline phosphatase enzyme, osteocalcin or osteopontin for bone, oil O red staining for lipid accumulation in MSCs that differentiate towards an adipocyte lineage.

The expanded MSCs of the invention can be analyzed based on gene expression profiles. In this manner, the multilineage commitment potential can be determined. As used herein, an “expression profile” comprises one or more values corresponding to a measurement of the relative abundance of a gene expression product. Such values may include measurements of RNA levels or protein abundance. Thus, the expression profile can comprise values representing the measurement of the transcriptional state or the translational state of the gene (see U.S. Pat. Nos. 6,040,138, 5,800,992, 6,020135, 6,344,316, and 6,033,860).

The transcriptional state of a sample includes the identities and relative abundance of the RNA species, especially mRNAs present in the sample. Preferably, a substantial fraction of all constituent RNA species in the sample are measured, but at least a sufficient fraction to characterize the transcriptional state of the sample is measured. The transcriptional state can be conveniently determined by measuring transcript abundance by any of several existing gene expression technologies. Translational state includes the identities and relative abundance of the constituent protein species in the sample. As is known to those of skill in the art, the transcriptional state and translational state are related.

In one embodiment of the invention, microarrays can be used to measure the values to be included in the expression profiles. Microarrays are particularly well suited for this purpose because of the reproducibility between different experiments. DNA microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of genes. Each array consists of a reproducible pattern of capture probes attached to a cell culture support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels (see U.S. Pat. Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and 6,344,316). High-density oligonucleotide arrays are particularly useful for determining the gene expression profile for a large number of RNAs in a sample.

“Array” is intended to mean a cell culture support or substrate with peptide or nucleic acid probes attached to the cell culture support or substrate. Arrays typically comprise a plurality of different nucleic acid or peptide capture probes that are coupled to a surface of a substrate in different, known locations. These arrays, also described as “microarrays” or colloquially “chips,” have been generally described in the art, for example, in U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186, 6,329,143, and 6,309,831 and Fodor et al. (1991) Science 251:767-77.

The methods of the invention comprise culturing or expanding MSCs from a stem cell source in a serum-free cell culture system that comprises a serum-free cell culture medium disclosed herein above, which comprises a cytokine cocktail and base medium. Methods of cell culture are well known in the art (see, e.g., Waymouth (1984) “Preparation and Use of Serum-free Culture Media,” in Cell Culture Methods for Molecular and Cell Biology, Vol. 1, Methods for Preparation of Media, Supplements, and Substrata for Serum-Free Animal Cell Culture, eds. Barnes et al. (Alan R. Liss), pp. 23-68). The term “cell-culture” is intended to mean the expansion or maintenance of cells in an artificial in vitro environment while maintaining a pluripotent phenotype. It is to be understood that the term “cell culture” is a generic term and may be used to encompass the cultivation not only of individual cells but also tissues, organ systems, or whole organisms.

In some embodiments, cells derived from a stem cell source are seeded onto a two-dimensional or three-dimensional cell culture surface described herein and contacted with the serum-free cell culture medium of the invention. The medium may initially contain the cytokine cocktail as well as the base medium, or the cytokine cocktail can be added later. Cells are then incubated with the serum-free cell culture medium comprising the cytokine cocktail and cell culture support at a temperature suitable for cell growth (for some embodiments about 37° C.), for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours, at least about 168 hours, at least about 192 hours, or longer. Cells may be harvested in any manner known in the art including but not limited to centrifugation after decanting non-adherent cells, trypsinizing adherent cells, or scraping cells from the surface of the cell culture support.

The MSCs used in culture can include cells derived from any stem cell source, such as umbilical cord, umbilical cord blood, placenta, embryonic stem cells, adipose tissue, bone marrow, or other tissue-specific mesenchyme. These samples may be fresh, frozen, or refrigerated. Methods of freezing cells are well known in the art (see, e.g., Doyle et al. (1995) Cell & Tissue Culture: Laboratory Procedures (John Wiley & Sons, Chichester)).

Cryopreservation of stem cells prior to culture or cryopreservation of expanded cells disclosed herein may be carried out according to known methods. For example, cells may be suspended in a “freezing medium” such as, for example, culture medium further comprising 10% dimethylsulfoxide (DMSO), with or without 5-10% glycerol, at a density, for example, of about 1-2×10⁶ cells/ml. The cells may be dispensed into glass or plastic vials, which are then sealed and transferred to a freezing chamber of a programmable or passive freezer. The optimal rate of freezing may be determined empirically. For example, a freezing program that gives a change in temperature of about −1° C./min through the heat of fusion may be used. Once vials containing the cells have reached −80° C., they may be transferred to a liquid nitrogen storage area. Cryopreserved cells may be stored for a period of years.

In some embodiments, freshly isolated cells from any stem cell source may be cryopreserved to constitute a bank of cells, portions of which may be withdrawn by thawing and then used to produce the expanded cells of the invention as needed. Thawing may be carried out rapidly, for example, by transferring a vial from liquid nitrogen to a 37° C. water bath. The thawed contents of the vial may be immediately transferred under sterile conditions to a culture vessel containing an appropriate medium such as nutritive medium. Once in culture, the cells may be examined daily, for example, with an inverted microscope to detect cell proliferation, and subcultured as soon as they reach an appropriate density.

Cells may be withdrawn from a cell bank as needed, and used for the production of new stem cells or tissue either in vitro, for example, as a three-dimensional scaffold culture, as described below, or in vivo, for example, by direct administration of cells to the site where tissue reconstitution or repair is needed. As described herein, the expanded MSCs of the invention may be used to reconstitute or repair tissue in a subject where the cells were originally isolated from that subject's own bone marrow or other tissue (i.e., autologous cells). Alternatively, the expanded MSCs disclosed herein may be used as ubiquitous donor cells to reconstitute or repair tissue in any subject (i.e., heterologous cells).

Methods of MSC isolation from bone marrow are well established. Prior to culture, a large proportion of non-mesenchymal linage cells may be removed from a stem cell source by negative or positive selection. For example, large numbers of lineage-committed cells can be removed by selective magnetic bead separations. In some embodiments, at least about 80%, usually at least about 70% of the differentiated cells will be removed prior to culture. Mononuclear cells from these tissues may be collected by density gradient centrifugation and cultured in tissue culture containers. Furthermore, bone marrow aspirates may be seeded onto tissue culture plates. Non-adherent cells may be removed after 1 to 3 days. Adherent, spindle shaped fibroblast-like MSCs are kept and expanded. After several hours to days, non-adherent cells are washed away and the MSCs remain. It has also been reported that MSCs may be obtained from cord blood, placenta and adipose tissues.

Cultured MSCs can be further isolated using any method known in the art. Generally, the MSCs are contacted with monoclonal antibodies directed to cell surface antigens and either positively or negatively selected. Such techniques for selection are well known in the art and include sorting by immunomagnetic beads, by complement mediated lysis, by “panning” with antibody attached to a solid matrix, agglutination methods, magnetic activated cell sorting (MACS), or fluorescence activated cell sorting (FACS).

The expanded MSCs of the invention have broad application in treating and ameliorating disease and injury. The expanded MSCs of the invention are useful in many therapeutic applications including repairing, reconstituting, and regenerating tissue as well as gene delivery. The MSCs of the invention can comprise both lineage-committed and uncommitted cells; thus, both cell types can be used together to accomplish multiple therapeutic goals, even simultaneously in some embodiments. For example, in some embodiments, the expanded MSCs of the invention can be used directly as stem cell transplants or be used in stem cell grafts either in suspension or on a cell culture support scaffold as noted herein above.

The expanded MSCs of the invention can be placed in a carrier medium before administration. For infusion, expanded MSCs of the invention can be administered in any physiologically acceptable medium, intravascularly, including intravenously, although they may also be introduced into other convenient sites such as into the bone marrow, where the cells may find an appropriate site for regeneration and differentiation. Usually, at least about 1×10⁵ cells/kg, at least about 5×10⁵ cells/kg, at least about 1×10⁶ cells/kg, at least about 2×10⁶ cells/kg, at least about 3×10⁶ cells/kg, at least about 4×10⁶ cells/kg, at least about 5×10⁶ cells/kg, at least about 6×10⁶ cells/kg, at least about 7×10⁶ cells/kg, at least about 8×10⁶ cells/kg, at least about 9×10⁶ cells/kg, at least about 10×10⁶ cells/kg, or more will be administered. See, for example, Ballen et al. (2001) Transplantation 7:635-645. The MSCs may be introduced by any method including injection, catheterization, or the like. If desired, additional drugs or growth factors can be co-administered. Drugs of interest include 5-fluorouracil and growth factors including cytokines such as IL-2, IL-3, G-CSF, M-CSF, GM-CSF, IFNγ, and erythropoietin. In addition, the MSCs can be injected with collagen, Matrigel, alone or with other hydrogels.

Administered MSCs may also comprise a mixture of cells herein described and additional cells of interest. Additional cells of interest include, without limitation, differentiated liver cells, differentiated cardiac muscle, differentiated pancreatic cells, and the like. These combinations are particularly useful when the expanded MSCs of the invention are seeded on a three-dimensional scaffold, hydrogel, or without carrier, as disclosed herein above.

In one embodiment, the expanded MSC population of the invention can be used to repair or reconstitute damaged or diseased mesenchymal tissues, such as the heart, the pancreas, the liver, fat tissue, bone, cartilage, endothelium, nerves, astrocytes, dermis, and the like. Once the expanded MSCs migrate to or are placed at the site of injury, they can differentiate to form new tissues and supplement organ function. In some embodiments, the cells are used to promote vascularization and, therefore, improve oxygenation and waste removal from tissues. In these embodiments, the expanded MSCs of the invention can be used to increase function of differentiated tissues and organs such as the ischemic heart as in cardiac failure or ischemic nerves as in stroke. Therefore, the expanded MSCs of the invention are useful in any disease where cellular function or organ function has been decremented.

“Supplementing a damaged organ” or “supplementing organ function” is intended to mean increasing, enhancing, or improving the function of an organ that is operating at less than optimum capacity. The term is used to refer to a gain in function so that the organ is operating at a physiologically acceptable capacity for that subject. For example, the physiological acceptable capacity for an organ from a child, e.g., a kidney or heart, would be different from the physiological acceptable capacity of an adult, or an elderly patient. The entire organ or part of the organ can be supplemented. Preferably the supplementation results in an organ with the same physiological response as a non-damaged or non-diseased organ. In one embodiment, an organ is supplemented in capacity when it is functioning to at least at about 10% of its natural capacity.

The expanded MSCs of the present invention can be used for implantation by contacting the cells with a tissue-engineered construct prior to grafting as noted herein above. The construct containing these cells is then implanted into a host in need of such a graft. The cells of the invention are particularly useful for promoting bone and cartilage generation, thereby facilitating tissue regeneration and repair. These cells may also be used in applications relating to graft versus host disease. The expanded MSCs of the invention may also be useful as vasculature-promoting stem cells. “Vascularization promoting” or “vasculature promoting” is intended to mean promoting the growth of new vessels (vasculogenesis) or inducing outgrowth from existing vessels (angiogenesis), or any combination thereof.

“Differentiated cells” is intended to mean cells that are committed to restricted tissue development. In some embodiments, the expanded cells of the invention may comprise both lineage-committed and uncommitted MSCs. Thus, in some tissue-engineered constructs, the MSCs may give rise to both the differentiated tissue and act as the source for the vasculature-promoting stem cells. In addition, in some tissue-engineered constructs, the MSCs may give rise to both the differentiated tissue and act as the source for the cardiovascular-promoting stem cells. In addition, in some tissue-engineered constructs, the MSCs may give rise to both the differentiated tissue and act as the source for the bone and cartilage-promoting stem cells. In other embodiments, the source of differentiated tissues may comprise cells or tissue from the intended graft recipient or another donor. The cell or tissue source may be differentiated prior to implantation. For example, pancreatic beta cells can be differentiated using conditions described for embryoid body formation as detailed in Itskovich-Eldor et al. (2000) Mol. Med. 6:88.

In one specific embodiment, the vasculature-promoting stem cells may be contacted with angiogenic growth factors such as VEGF and bFGF. These stem cells may be contacted with angiogenic growth factors prior to or after seeding onto the scaffold before engrafting the tissue-engineered construct into a damaged organ. In some embodiments, cytokine-impregnated polymers can release the angiogenic growth factor, such as VEGF and bFGF, over time. In other embodiments, the scaffold may be programmed to drive expansion and proliferation of seeded cells (see, e.g., U.S. Patent Application Publication No. 20040063206). In other embodiments, microspheres or microcarriers may be contacted with the vasculature-promoting stem cells and placed at a target site. Microsphere-based scaffolds are well known in the art (see, e.g., Mahoney and Saltzman (2001) Nature Biotech. 19:934).

In one specific embodiment, the cardiovascular- or bone and cartilage-promoting stem cells may be contacted with growth factors such as BDNF, bFGF, BMP-2, BMP-4, DKK-1, EGF, EPO, fibronectin, Flt-3/Flk-2 ligand, G-CSF, IGF-1, IL-11, IL-3, IL-5, IL-6, LIF, PD-ECGF, SCF, SDF 1-α, TGF-β, thrombospondin, and WNT signaling agonists, including but not limited to BIO and WNT proteins, WNT-1, WNT-2, WNT-2b, WNT-3, WNT-3a, WNT-4, WNT-5a, WNT-5b, WNT-6, WNT-7a, WNT-7b, WNT-8a, WNT-8b, WNT-9a, WNT-9b, WNT-10a, WNT-10b, WNT-11, and WNT-16. In one embodiment, the WNT protein is WNT-3a.

The expanded MSCs of the invention can also be used for gene therapy in patients in need thereof. In some embodiments, more mature lineage-committed cells will be useful, especially where transient gene expression is needed or where gene transduction is facilitated by the maturation and division of the cells. For example, some retroviral vectors require that the cell be cycling for the gene to be integrated. Methods for transducing stem and progenitor cells to deliver new and therapeutic genes are known in the art.

Another aspect of the invention is a kit useful for promoting the attachment, survival, and/or proliferation of MSCs, comprising a serum-free cell culture system of the invention is provided. Such a kit comprises a serum-free cell culture medium and a cell culture surface described herein, and can comprise one or more other components or reagents suitable for culturing the cell. In one embodiment, the serum-free cell culture medium comprises any of the cytokine cocktails described herein, for example, the cocktail shown in Table 1 below, and a base medium, for example, the base medium shown in Table 2 below, and a cell culture surface comprising a cell culture support bound to a CAR, which in turn is bound to an ECM protein. In some embodiments, the ECM is a mixture of collagen I and fibronectin. In other embodiments, the kits comprise these components and also comprise one or more reagents for measuring cell proliferation in the culture. Such kits have many uses, which will be evident to one of skill in the art. For example, they can be used to propagate MSCs to be used in methods of cell therapy. Such kits could be of commercial use, e.g., in high-throughput drug studies.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

For these examples, the concentrations of various cytokines in the culture media are described in Table 1, while the base medium composition is described in Table 2. TABLE 1 Cytokine Cocktail for Serum-Free Cell Culture Medium Concentration Growth Factor Abbrev. Factor Full Name Vendor (ng/ml) BDNF Brain-derived Neurotrophic Factor R&D Systems, MN 1 bFGF Basic Fibroblast Growth Factor BD Biosciences, MA 2.5 BIO (2′Z,3′E)-6-bromoindirubin-3′-oxime Calbiochem 50 nM BMP-2 Bone Morphogenic Protein 2 R&D Systems, MN 0.5 BMP-4 Bone Morphogenic Protein 4 R&D Systems, MN 0.5 DKK-1 Dickkopf 1 R&D Systems, MN 10 EGF Epidermal Growth Factor BD Biosciences, MA 10 Erythropoietin Erythropoietin R&D Systems, MN .05 units/ml Fibronectin Fibronectin BD Biosciences, MA 10 Flt-3/Flk-2 ligand Flt-3/Flk-2 ligand R&D Systems, MN 5 G-CSF Granulocytes colony stimulating factor R&D Systems, MN 1 IGF-1 Insulin-like Growth Factor 1 R&D Systems, MN 2.5 IL-11 Interleukin-11 R&D Systems, MN 0.1 IL-3 Interleukin-3 R&D Systems, MN 1 IL-5 Interleukin-5 R&D Systems, MN 0.1 IL-6 Interleukin-6 R&D Systems, MN 0.2 LIF Leukemia Inhibitory Factor Chemicon 10 PD-ECGF Platelet-derived Endothelial Cell Growth Factor R&D Systems, MN 2 SCF Stem Cell Factor BD Biosciences, MA 2 SDF-1 α Stromal Cell Derived Factor 1 α R&D Systems, MN 3 TGF-β Transforming Growth Factor-β BD Biosciences 0.1-1 Thrombospondin Thrombospondin Sigma-Aldrich, MO 10 WNT-3a Wnt protein 3a R&D Systems, MN 10

TABLE 2 Base Medium for Serum-Free Cell Culture Medium Base Medium Component Amount DMEM/MCDB-201 Media 60:40 Ascorbic acid 2-phosphate 1 × 10⁻⁴ M Dexamethasone 1 × 10⁻⁹ M Linoleic Acid-Bovine Serum 0.1% Albumin Insulin 5 μg/mL Transferrin 5 μg/mL Sodium selenite 5 ng/mL

Example 1 MSC Expansion in Various Serum-Free Media

MSCs along with complete growth medium were purchased from Cambrex Biosciences (Baltimore, Md.). Frozen cells were thawed and cultured following manufacturer's instruction. After reaching ˜90% confluency, MSCs were washed once with PBS, removed from the culture surface using Trypsin/EDTA and replated at a density of 1600 cells/well (for 96 well plates) or at a density of 50,000 cells/well (for 6-well plates) which corresponds to the manufacturers recommended seeding density of 5000 cells/cm².

At day 6 or 7, cells were washed once with PBS, fixed using 4% paraformaldehyde for 15 minutes and then stained using DAPI according to manufacturer's suggestions (Molecular Probes, Eugene, Oreg.). For 96-well plate experiments, one image per well was taken at 4× magnification using Molecular Devices' Discovery-1 high content screening system. Cell nuclei enumeration was determined using Metamorph Image Analysis Software (Molecular Devices, Sunnyvale, Calif.). The number of DAPI stained cell nuclei corresponds directly with the total number of cells per well. For 6-well plates, cell enumeration was determined using Trypan Blue exclusion and manual counting using a hemocytometer. Multiple donors (different preps of MSCs) were tested in serum-free conditions while the cells were also cultured on tissue culture polystyrene (TCPS) plates using the manufacturer's standard serum-containing medium as the control.

Several serum-free expansion conditions were scaled up into the 6-well plate format (˜10 cm²) and cultured for 7 days. Cells were then washed with PBS and then removed from the 6-well plates using Trypsin/EDTA. Cells were then reseeded into 96-well plates at two different densities according to manufacturer's specifications. MSCs induced to form adipocytes were seeded at 7000 cells/well and cultured in Stem Cell Technologies (Vancouver, B. C., Canada) adipogenic medium supplement. MSCs induced to form osteoblasts were seeded at 1000 cells/well using Cambrex Biosciences (Baltimore, Md.) osteogenic media supplements. Two to three weeks after induction, cells were washed once with PBS and fixed using 4% paraformaldehyde. Fixed cells were then stained with Oil Red O (Sigma-Aldrich (St. Louis, Mo.)) for adipocytes and BCIP/NBT (Sigma-Aldrich (St. Louis, Mo.)) for alkaline phosphatase/osteoblasts. Bright field images at multiple sites per well were taken of stained cells at 10× magnification. Percentage of differentiation was determined using Metamorph Image Analysis Software (Molecular Devices, Sunnyvale, Calif.) to calculate total stained area by threshold image analysis.

Initial experiments compared serum-free medium expansion of MSCs in G2 medium on Collagen I (Col 1, Sigma-Aldrich)/Fibronectin (FN, BD Biosciences) cell adhesion resistant (CAR) surface modified 96-well plates. MSCs were expanded for 7 days in G2 serum-free medium on the CAR surface and cell nuclei counts were compared to MSCs expanded in the manufacturer's recommended serum-containing medium on tissue culture polystyrene (TCPS) 96-well plates. MSCs expanded in the G2-CAR surface serum free environment were comparable to the manufacturer's recommended serum-containing expansion conditions up to 7 days (see FIG. 1).

Example 2 Differentiation Capacity and Surface Marker Characterization of G2 Expanded MSCs

MSCs expanded in serum-free media of the present invention on CAR Col 1/FN surface for 7 days remained multipotent. Briefly, MSCs expanded in culture environments of the present invention were removed from the serum-free media and then cultured in either adipogenic (fat) or osteogenic (bone) induction media (per manufacturer's suggestions). MSCs expanded in the serum-free environments of the present invention were able to differentiate towards both adipogenic (see FIG. 2) and osteogenic (see FIG. 3) lineages. These results demonstrate that the serum-free environments of the present invention help to maintain stem cell pluripotency in expanded MSCs at least as well as the industry standard serum-containing media (Cambrex). This demonstrates that these cells are not committed towards specific lineages, and that therefore these MSCs may still be used for a variety or research or clinical applications.

MSCs cultured in serum-free environments for 7 days retain cell surface markers showing equivalency to Cambrex cell populations expanded in serum-containing media. In addition, MSCs cultured in serum-free environments lacked CD45 marker expression, indicating that these cells do not differentiate towards hematopoietic lineages.

Example 3 Comparison of Media and Cell Culture Surface Conditions

MSCs cultured in three different media (G2, base medium without G2 growth factors/cytokines, and in serum-containing complete medium) all expand better on a Col 1/FN CAR surface as compared to TCPS surfaces (see FIG. 4). Also, MSCs cultured in G2 serum-free medium expand to equivalent levels as MSCs cultured in serum-containing medium.

Example 4 Media Optimization

Media compositions that support the expansion of MSCs were identified. The G2 serum-free medium served as a control for this optimization experiment. Screening plates were prepared using 96-well plates containing 30 factors in 60 different compositions. One thousand six hundred (1600) MSCs were incubated in each well for 7 days. Best media compositions were selected based on the average number of live cells obtained from the repeats from each screen.

Best well “hits” of the cytokine screen were the C6, D3, C2, and G5 wells, respectively.

The C6 well contained cytokines BDNF, bFGF, BIO, BMP-2, BMP-4, DKK-1, EGF, EPO, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, G-CSF, IGF-1, IL-11, IL-3, IL-5, IL-6, LIF, PD-ECGF, SCF, SDF-1α, and WNT-3a.

The D3 well contained cytokines bFGF, BMP-2, EGF, EPO, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and WNT-3a

The C2 well contained cytokines bFGF, BIO, BMP-2, BMP-4, EGF, EPO, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and WNT-3a.

The G5 well contained cytokines bFGF, BMP-4, DKK-1, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-6, PD-ECGF, SDF-1α, and WNT-3a.

Cytokine compositions of wells C6, D3, C2, and G5 are referred to hereinafter as “C6,” “D3,” “C2,” and “G5,” respectively.

Media compositions C6, D3, C2, and G5 all outperformed G2 serum free medium controls and 10% serum-containing complete medium (see FIG. 5). These serum free media compositions contain 22, 11, 11, and 10 growth factor/cytokine additives, respectively. In addition, serum free media G2 and D3 both performed as well or better than serum-containing complete medium controls and significantly outperform a previously published serum free MSC expansion medium (Lennon et al. (1995) Exp. Cell Res. 219:211-222; U.S. Pat. No. 5,908,782).

Three of these serum-free media compositions (D3, C2, and G5) were used to confirm MSCs retained multipotentiality. MSCs were cultured in one of the three serum-free media for 7 days. Expanded MSCs were removed from the serum-free media and then cultured in either adipogenic (fat) or osteogenic (bone) induction media (per manufacturer's suggestions). MSCs remained multipotential and differentiated towards both adipogenic and osteogenic lineages in all three serum-free media, each comparable to the manufacturer's recommended serum-containing complete medium controls (see FIGS. 6A and 6B).

Further media optimization was performed and the best well “hits” from this cytokine screen were the G7, G4 and C8 wells, respectively.

The G7 well contained cytokines bFGF, BMP-2, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, LIF, and WNT-3a.

The G4 well contained cytokines bFGF, BMP-2, EGF, IL-11, PD-ECGF, and WNT-3a.

The C8 well contained cytokines bFGF, BMP-2, EGF, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, IL-5, LIF, PD-ECGF, and WNT-3a.

Cytokine compositions of wells G7, G4, and C8 are referred to hereinafter as “G7,” “G4,” and “C8,” respectively. Media compositions G7, G4, and C8 all expanded MSCs as well as or better than serum-containing controls (see FIG. 7).

Additional optimization data was collected employing a variety of media compositions. In one experiment, MSCs were cultured in: 1) the G4 serum-free medium composition; 2) G4+TGF-β; 3) bFGF in base medium only; 4) EGF in base medium only; 5) TGF-β in base medium only; 6) base medium alone; and 7) serum-containing medium. Although MSCs cultured in G4 expanded as well or better than serum-containing medium, MSCs cultured in G4+TGF-β far outperformed any other media composition, nearly triple the serum-containing medium controls (see FIG. 8). Furthermore, the single factors, while promoting MSC expansion, did not perform as well as serum in expanding MSCs. However, synergistic combinations of these factors (e.g., G4) expand MSCs more than any single factor, and the addition of TGF-β substantially enhanced expansion. These factor synergies, in combination with the collagen 1+fibronectin surface, comprise serum-free environments that substantially outperform anything that has been established to date. G4+TGF-β serum-free medium was scaled up and used to expand MSCs in 6-well Col 1/FN CAR plates. MSCs were seeded on 6-well plates at 50,000 per well (n=3). Cells in serum containing complete media were seeded onto a 6-well TCPS plate. At confluency, all cells were removed and cell number was determined using Trypan Blue exclusion and hemocytometer counting. Cells were replated at 50,000 per well under the same condition for a second round of expansion. Cell numbers were again determined as above. After the first round of expansion (approximately 6 days), MSCs expanded in serum-free conditions outperformed the manufacturer's recommended serum-containing medium controls. After the second round of expansion (approximately 6 days), the MSCs continued to expand in the serum-free medium at an increased rate as compared to the serum-containing medium controls. G4+TGFb provided 5.4 and 6.5 fold expansion in the first and second rounds, respectively, and regular medium gave 3.5 and 3.1 fold expansion in the first and second rounds, respectively.

In another experiment, a serum-free medium comprising base medium and WNT-3a, bFGF, and TGF-β was compared to media compositions containing the same components but adding one, two, three, four, five, six, or seven additional cytokines that include EGF, fibronectin, thrombospondin, BMP-2, BMP-4, IL-11, and PD-ECGF in their ability to expand MSCs in culture. Adding one, two, three, four, five, six, or seven growth factors to the serum free medium comprising base medium and WNT-3a, bFGF, and TGF-β had a positive effect on MSC expansion. In particular, the addition of EGF to the three growth factors already present in the serum-free medium resulted in approximately a 20% increase in MSC expansion over compositions that did not contain EGF, and both BMPs on top of EGF further enhanced expansion. Furthermore, the addition of all seven cytokines to WNT-3a, bFGF, and TGF-β gave the best expansion, demonstrating that building complexity into the serum free environments may further enhance MSC expansion. However, the synergies between WNT-3a, bFGF, EGF, and TGF-β were found to have the greatest effects on MSC serum-free expansion on collagen 1+fibronectin surfaces.

In another experiment, a serum-free medium comprising base medium was compared to media comprising the base medium plus various combinations of WNT-3a, BIO, bFGF, EGF, and TGF-β in their ability to expand MSCs in culture (see FIG. 9). Specifically, the combinations tested were: base medium alone (no growth factors), Cambrex Biosciences (Baltimore, Md.) serum-containing medium on TCPS, base medium+bFGF+TGF-β, base medium+WNT-3a+bFGF, base medium+bFGF+EGF, base medium+WNT-3a+TGF-β, base medium+WNT-3a+EGF, base medium+EGF+TGF-β, base medium+WNT-3a, base medium+bFGF+EGF+TGF-β, base medium+WNT-3a+bFGF+EGF+TGF-β, and base medium+bFGF+EGF+TGF-β+BIO. Although all conditions outperformed base medium alone, the growth factor combinations that produced the greatest MSC expansion were base medium+EGF+TGF-β, base medium+bFGF+TGF-β, base medium+bFGF+EGF+TGF-β, base medium+WNT-3a+bFGF+EGF+TGF-β, and base medium+bFGF+EGF+TGF-β+BIO.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. 

1. A serum-free cell culture system comprising a serum-free cell culture medium and a cell culture surface that promotes the adhesion and expansion of mesenchymal stem cells, wherein at least one insoluble substrate protein is presented from said cell culture surface, and wherein said serum-free cell culture medium is selected from the group consisting of: a) a medium comprising bFGF in combination with at least one growth factor selected from the group consisting of a WNT signaling agonist, TGF-β, and EGF; b) a medium comprising a WNT signaling agonist in combination with at least one growth factor selected from the group consisting of bFGF, TGF-β, and EGF; c) a medium comprising TGF-β in combination with at least one growth factor selected from the group consisting of bFGF, a WNT signaling agonist, and EGF; d) a medium comprising EGF in combination with at least one growth factor selected from the group consisting of bFGF, a WNT signaling agonist, and TGF-β; e) a medium comprising fibronectin, SDF-1 α, IL-6, SCF, IL-5, BDNF, PD-ECGF, IL-11, IL-3, EPO, Flt-3/Flk-2 ligand, BMP-4, thrombospondin, IGF-1, and bFGF; f) a medium comprising BDNF, bFGF, BIO, BMP-2, BMP-4, DKK-1, EGF, EPO, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, G-CSF, IGF-1, IL-11, IL-3, IL-5, IL-6, LIF, PD-ECGF, SCF, SDF-1α, and a WNT signaling agonist; g) a medium comprising bFGF, BMP-2, EGF, EPO, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and a WNT signaling agonist; h) a medium comprising bFGF, BIO, BMP-2, BMP-4, EGF, EPO, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and a WNT signaling agonist; i) a medium comprising bFGF, BMP-4, DKK-1, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-6, PD-ECGF, SDF-1α, and a WNT signaling agonist; j) a medium comprising bFGF, BMP-2, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, LIF, and a WNT signaling agonist; k) a medium comprising bFGF, BMP-2, EGF, IL-11, PD-ECGF, and a WNT signaling agonist; and l) a medium comprising bFGF, BMP-2, EGF, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, IL-5, LIF, PD-ECGF, and a WNT signaling agonist.
 2. The serum-free cell culture system of claim 1, wherein said WNT signaling agonist is BIO or WNT-3a.
 3. The serum-free cell culture system of claim 1, wherein the insoluble substrate protein is an extracellular matrix protein.
 4. The serum-free cell culture system of claim 3, wherein the extracellular matrix protein is fibronectin, laminin, HA, vitronectin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or any combination thereof.
 5. The serum-free cell culture system of claim 4, wherein the extracellular matrix protein is a combination of collagen I and fibronectin.
 6. The serum-free cell culture system of claim 5, wherein the WNT signaling agonist is BIO or WNT-3a, and wherein the cell culture surface comprises a cell culture support bound to a cell adhesion resistant material.
 7. A method of expanding a population of mesenchymal stem cells using the cell culture system of claim
 1. 8. The method of claim 7, wherein said WNT signaling agonist is BIO or WNT-3a, wherein the cell culture surface comprises a cell culture support bound to a cell adhesion resistant material, and wherein the insoluble substrate protein is a combination of collagen I and fibronectin.
 9. An expanded population of mesenchymal stem cells that has never been exposed to serum, wherein said population is produced by the method of claim
 7. 10. A method of transplanting an expanded population of mesenchymal stem cells, comprising administering the population of claim 9 to a patient in need thereof, wherein the population is administered in an amount effective to reconstitute cardiovascular, bone, or cartilage tissue.
 11. A kit comprising the serum-free cell culture system of claim
 1. 12. The kit of claim 11, wherein said WNT signaling agonist is BIO or WNT-3a, wherein the cell culture surface comprises a cell culture support bound to a cell adhesion resistant material, and wherein the insoluble substrate protein is a combination of collagen I and fibronectin.
 13. A serum-free cell culture system comprising a serum-free cell culture medium and a cell culture surface, wherein said cell culture surface promotes the adhesion and expansion of mesenchymal stem cells and comprises a cell culture support bound to a cell adhesion resistant material, and wherein at least one insoluble substrate protein is presented from the cell culture surface.
 14. The serum-free cell culture system of claim 13, wherein the insoluble substrate protein is an extracellular matrix protein.
 15. The serum-free cell culture system of claim 14, wherein the extracellular matrix protein is fibronectin, laminin, HA, vitronectin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or any combination thereof.
 16. The serum-free cell culture system of claim 15, wherein the extracellular matrix protein is a combination of collagen I and fibronectin.
 17. The serum-free cell culture system of claim 13, wherein said serum-free cell culture medium is selected from the group consisting of: a) a medium comprising bFGF in combination with at least one growth factor selected from the group consisting of a WNT signaling agonist, TGF-β, and EGF; b) a medium comprising a WNT signaling agonist in combination with at least one growth factor selected from the group consisting of bFGF, TGF-β, and EGF; c) a medium comprising TGF-β in combination with at least one growth factor selected from the group consisting of bFGF, a WNT signaling agonist, and EGF; d) a medium comprising EGF in combination with at least one growth factor selected from the group consisting of bFGF, a WNT signaling agonist, and TGF-β; e) a medium comprising fibronectin, SDF-1 α, IL-6, SCF, IL-5, BDNF, PD-ECGF, IL-11, IL-3, EPO, Flt-3/Flk-2 ligand, BMP-4, thrombospondin, IGF-1, and bFGF; f) a medium comprising BDNF, bFGF, BIO, BMP-2, BMP-4, DKK-1, EGF, EPO, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, G-CSF, IGF-1, IL-11, IL-3, IL-5, IL-6, LIF, PD-ECGF, SCF, SDF-1α, and a WNT signaling agonist; g) a medium comprising bFGF, BMP-2, EGF, EPO, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and a WNT signaling agonist; h) a medium comprising bFGF, BIO, BMP-2, BMP-4, EGF, EPO, Flt-3/Flk-2 ligand, IGF-1, IL-11, IL-5, and a WNT signaling agonist; i) a medium comprising bFGF, BMP-4, DKK-1, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-6, PD-ECGF, SDF-1α, and a WNT signaling agonist; j) a medium comprising bFGF, BMP-2, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, LIF, and a WNT signaling agonist; k) a medium comprising bFGF, BMP-2, EGF, IL-11, PD-ECGF, and a WNT signaling agonist; and l) a medium comprising bFGF, BMP-2, EGF, fibronectin, thrombospondin, Flt-3/Flk-2 ligand, IL-11, IL-5, LIF, PD-ECGF, and a WNT signaling agonist.
 18. The serum-free cell culture system of claim 17, wherein said WNT signaling agonist is BIO or WNT-3a.
 19. The serum-free cell culture system of claim 17, wherein the insoluble substrate protein is an extracellular matrix protein and wherein the extracellular matrix protein is fibronectin, laminin, HA, vitronectin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, or any combination thereof.
 20. The serum-free cell culture system of claim 19, wherein the extracellular matrix protein is a combination of collagen I and fibronectin.
 21. A method of expanding a population of mesenchymal stem cells using the cell culture system of claim
 13. 22. The method of claim 21, wherein the insoluble substrate protein is a combination of collagen I and fibronectin.
 23. An expanded population of mesenchymal stem cells, said population produced by the method of claim 21, wherein said cells were unexposed to serum.
 24. A method of transplanting an expanded population of mesenchymal stem cells, comprising administering the population of claim 23 to a patient in need thereof, wherein the population is administered in an amount effective to reconstitute cardiovascular, bone, or cartilage tissue.
 25. A kit comprising the serum-free cell culture system of claim
 13. 26. The kit of claim 25, wherein the insoluble substrate protein is a combination of collagen I and fibronectin. 